1. Field of the Invention
This invention is related in general to the field of electrophoretic analysis of liquid samples. In particular, the invention relates to an apparatus and method for the electrophoresis process beginning with the step of applying liquid samples to an electrophoresis support media and, without moving the support media, further including the steps of electrophoresing, staining, incubating, and drying the sample. The present invention further includes the scanning and densitometry measurements on the scanned samples in both the visible and fluorescent modes.
Electrophoresis is the science of moving charged particles in an electric field through a solid or semi-solid media. The technique is most commonly used in medical research in medical laboratories for analyzing various blood proteins.
2. Description of the Prior Art
In the diagnoses of ailments of human beings and animals, it is known that much information can be provided by an analysis of specific biological fluids such as blood serum proteins, lipoproteins, hemoglobin, and isoenzymes. It is well known that electrophoresis is an effective method of separating the respective components of such fluids for analysis or for employing optical densitometry techniques in analyzing the samples.
In the basic method of electrophoresis, charged molecules of the sample fluids are separated under the influence of an electrical field wherein the liquid sample to be examined is applied to a support medium having a porous surface. Because the various components of the fluid move at different rates through the support medium, the liquid sample may be separated into its respective components. Subsequent staining of the fractional components in the support medium may then be subjected to optical densitometry or other methods for examination.
The electrophoresis process has been performed through a series of manual steps for many years. The manual process typically has started with the laboratory technician preparing an electrophoresis chamber by filling appropriate cavities of the chamber with buffer solution. Buffer solution is a liquid used in the electrophoresis process to maintain the support medium surface in a moist condition and to provide an electrical interface to a power source applied to the chamber so that an electric field may be applied to the support medium. The support medium is typically a piece of MYLAR.TM. backing which has been coated with a gel substance such as cellulose acetate or agarose. The liquid sample to be examined is typically a blood serum, but of course may be other liquids, the components of which may be moved through an electric field.
After the laboratory technician has prepared the electrophoresis chamber, the laboratory technician applies as precisely as possible, consistent volumes of the samples to precise locations on the support medium. The laboratory technician then places the support medium into the electrophoresis chamber so that the edges of the support medium are immersed in two buffer cavities at each of its longitudinal ends. Electrophoresis is then performed using a precise and consistent high voltage applied for a precise and consistent interval of time across the buffer cavities.
After electrophoresis has been completed, the laboratory technician applies a uniform coating of staining reagent or stain to the surface of the support medium allowing a precise and consistent interval of time for the reagent and sample to chemically combine. The staining reagent is a liquid used after electrophoresis to chemically combine with the separated components of the fluid sample, causing its components to exhibit optical characteristics.
Next, the laboratory technician incubates the support medium using a precise and consistent temperature and time interval. Incubation is the process of controlling the chemical reaction between the components of the liquid sample and the staining reagent by means of applying heat for a fixed interval of time.
Next, the laboratory technician dries the sample plate by increasing the temperature for a second precise and consistent temperature and time interval. The drying process stops the reaction between the sample plate and the reagent by removing water from the support medium.
One of the problems associated with the manual support medium preparation relates to the difficulty of precise application of the liquid samples to the support medium which is to be subjected to electrophoresis. The samples may be applied to the support medium one at a time in serial fashion with a hand pipettor, but the hand pipettor must be rinsed with a cleansing agent and blotted before a new sample is aspirated and then applied to the strip. Applicators have been designed to apply fluid samples simultaneously or in "parallel" to the strips. Such applicators are described at page 61 of the General Products Catalog for 1984-1985 of Helena Laboratories of Beaumont, Tex. Such applicators may apply eight, twelve or more samples to a microporous support medium and have the advantage of making the electrophoresis technique easier and more reproducible.
There have been prior art apparatus and method available for automatically performing electrophoresis and staining of the plurality of samples applied to a support medium. For example, U.S. Pat. No. 4,360,418 to Golias and U.S. Pat No. 4,391,689 to Golias describe an automated electrophoresis and staining apparatus and method.
Such apparatus includes an electrophoresis chamber and a series of vats mounted upon a platform and arranged in a row where the vats are adapted to contain respectively a liquid stain and a series of plate processing solutions. A plate holder rack, having a horizontal open frame, supports an upright electrophoresis plate or support medium onto which a sample for electrophoretic fractionization has been applied. Such electrophoresis plates required the initial preparation by application of liquid samples either manually or by using one of the parallel applicators described above. The plate is nested within a chamber which includes an electrophoretic circuit and the samples are electrophoresed for a predetermined time period. A power operated lift and transfer assembly is provided to lift, transfer and lower the plate holder rack and plate from the chamber progressively into each of the underlying vats for a predetermined period in a linear stepping motion while maintaining the plate in an upright position at all times. It is noted that the staining process relies on chemical procedures for the staining process rather than the manual system described above where incubation and drying are used. Although the apparatus described above has many desirable features, it has a practical disadvantageous feature in that it requires providing a plurality of chemicals and wash solutions in the unit which must be maintained periodically.
Prior art apparatus and methods for optically scanning support media which have been subjected to electrophoresis and staining have used devices such as photomultipler tubes, photodiodes or similar devices which produce an electric current or voltage output proportional to the light falling on such device. These devices are generally referred to as detectors. Prior art instruments employing these detectors are used for determining various physical properties of the samples which have been prepared by electrophoresis. The properties of interest concerning the separated bands of the sample are size and optical density or intensity of emitted light which is of a wave length different from that of the excitation light source. Separated bands of each sample which have been subjected to electrophoresis are known components of the sample under test and it is desirous that they be quantified for the purpose of aiding in medical diagnosis or research.
The known instruments which use the detectors referred to above generally find it necessary to use a blocking optical slit. The purpose of the slit is to allow the detector to "instantaneously view" a portion of the sample plate which is the same relative size and shape as the slit. The detector then produces an electrical current or voltage which is proportional in amplitude to the magnitude of the light detected. The current or voltage produced is then converted by means of an analog to digital converter and the resultant digital representation of the light magnitude is stored in an organized format in a digital computer memory.
It is an object of the present invention to provide automatic electrophoresis through the drying step, i.e., all the steps being performed without laboratory technician intervention.
It is an object of the present invention to provide automatic electrophoresis with fluorescent scanning, i.e., all the steps through and including the fluorescent scanning being performed without laboratory technician intervention.
It is an object of the present invention to provide an electrophoresis system with both visual and fluorescent scanning modes.
It is an object of the present invention to provide in-situ fluorescent scanning.
It is an object of the present invention to automatically deposit a sample in any location or configuration on the electrophoresis plate.
It is an object of the present invention to vary the quantity of the sample drawn from a sample supply and delivered to the electrophoresis plate.
It is an object of the present invention to aspirate and to deposit the sample from various position along the Z-axis (vertical).
It is an object of the present invention to control temperature within an electrophoresis chamber.
It is an object of the present invention to provide a thermoelectric heating/cooling device in the bottom of the electrophoresis chamber for cooling the electrophoresis plate.
It is an object of the present invention to provide additional electrodes to create a plurality of electrical zones within the electrophoresis chamber.
It is an object of the present invention to provide automatic electrophoresis with fluorescence with self-calibration.
It is an object of the present invention to automatically edit the graphic results obtained by scanning the electrophoresed sample to exclude aberrational data, including noise and artifacts.